Peripheral neuropathy is an established pain disorder (Hsiao et al., 2010; St Sauver et al., 2013) that encompasses multiple types varying in their clinical presentation, severity of pain, reversibility of neuronal damage, and prognosis (Stubblefield et al., 2009). The clinical presentation of peripheral neuropathy involves a wide range of signs and symptoms that largely depend on the etiology and site of neuronal damage. The etiology of peripheral neuropathy includes genetic, autoimmune, infectious, nutritional, and metabolic imbalances. In addition, peripheral neuropathy develops as a consequence to the exposure to anticancer chemotherapeutic treatment which is often referred to as chemotherapy-induced peripheral neuropathy (CIPN). The duration, mechanism, and onset of CIPN are unpredictable, but could be linked to factors such as age, type of chemotherapy, and extent of drug exposure. Importantly, CIPN may interfere with drug dosing, patient’s compliance, and patient’s lifestyle, which, in many cases, can lead to the discontinuation of the treatment (Colvin, 2019). Although many patients with peripheral neuropathy may improve after cessation of chemotherapy, some patients’ symptoms may persist or worsen due to persistent nerve damage (Cavaletti and Marmiroli, 2010). Unfortunately, multiple treatment modalities have been suggested to manage CIPN symptoms, but no effective strategy has been yet adopted (Flatters et al., 2017).

The prototypical platinum compound, cisplatin, which is still widely used for the treatment of various malignancies, results in sensory peripheral neuropathy with a typical sock-and-glove presentation (Staff et al., 2019). Other chemotherapies that also have been shown to precipitate CIPN include microtubule poisons, bortezomib, and thalidomide (Winer et al., 2004; Jones et al., 2005; Cavaletti and Marmiroli, 2010; Richardson et al., 2012). While the ability of these drugs to cause neuropathy is established, the pathophysiology of CIPN remains largely undetermined. Some studies indicated that taxanes disrupt the neuronal microtubules and deplete axonal energy supply due to mitochondrial damage (Argyriou et al., 2012; Miltenburg and Boogerd, 2014). Other evidence has also suggested that vinca alkaloids-induced peripheral neurotoxicity occurs as a consequence to structural and functional alterations of the cytoskeleton (Argyriou et al., 2012; Miltenburg and Boogerd, 2014). On the other hand, platinum compounds toxicity was thought to be promoted through neuronal mitochondrial dysfunction and apoptosis (Kim et al., 2015). This is largely based on the ability of cisplatin to form platinum-DNA adducts in the neurons of the dorsal root ganglia (DRG) (Kim et al., 2015). Moreover, cisplatin was shown to induce alterations in calcium signaling and homeostasis, thus altering neuroexcitability of DRG neurons (Erol et al., 2016). Similar to taxanes, cisplatin can also interact with components of the cytoskeletal microtubules and result in dysfunctional neuronal transport (Tulub and Stefanov, 2001). Importantly, a large body of evidence has suggested that CIPN is mediated through an inflammatory process that is mediated by the increased production of a spectrum of pro-inflammatory cytokines and chemokines (Brandolini et al., 2019). The contribution of inflammation to the pathogenesis of neuropathy has suggested the use of anti-inflammatory compounds and immunomodulators for the treatment of CIPN (Hu et al., 2018).

Rapamycin is a well-known anticancer and immunosuppressive agent that has been found to possess neuroprotective properties (Okamoto et al., 2016; Singh et al., 2017). The anticancer mechanism of action of rapamycin is based on its ability to inhibit the mammalian target of rapamycin (mTOR), the main player in a pathway that is dysregulated in several types of malignancies (Mita et al., 2003; Bjornsti and Houghton, 2004; Majumder et al., 2004). Moreover, adding rapamycin to chemotherapeutic regimens reduced the extent of chemotherapeutic resistance in tumor cells (Guri and Hall, 2016). Furthermore, several reports in the literature revealed that rapamycin has a protective effect against many neurodegenerative processes implicated in parkinsonism both in vitro and in vivo (Pan et al., 2008; Tain et al., 2009; Malagelada et al., 2010). Other studies have shown that rapamycin treatment reduced the progression of spinal cord-induced neuropathic pain in mice (Tateda et al., 2017). Interestingly, semisynthetic analogs of rapamycin significantly promoted neurite survival and neuronal proliferation, indicating that rapamycin itself, as well as its analogs, can be used as neuroprotective agents (Ruan et al., 2008). Nevertheless, rapamycin’s effect in mitigating symptoms of CIPN has not yet been investigated in preclinical models.

In this work, we aimed to investigate the potential of a well-established immunomodulator to exert a neuroprotective effect to alleviate the neurotoxicity induced by cisplatin in vivo (Ta et al., 2009). We also conducted additional in vitro experiments to understand the underlying cellular mechanisms of cisplatin-induced neurotoxicity and rapamycin neuroprotective function (Lapresa et al., 2019). Prevention of cisplatin-induced peripheral neuropathy by rapamycin might improve cancer patients’ quality of life and increases patients’ compliance.

The development of peripheral neuropathy is a treatment-limiting adverse effect of cancer therapy. CIPN may impinge on optimal drug dosing, patient’s compliance to treatment, and patient’s lifestyle, which collectively can lead to a premature discontinuation of chemotherapeutic treatment. The current major challenge in CIPN research include identifying effective pharmacological therapy that will both mitigate symptoms and allow for the completion of anticancer treatment. Recent evidence have shown that different compounds have neuroprotective properties and demonstrated promising effects in reducing CIPN in preclinical models (Galley et al., 2017; Kyte et al., 2018). However, these studies do not confirm whether these agents would prevent tumor progression or interfere with the antitumor potential of chemotherapy. For example, nicotine, which was shown to be effective in alleviating CIPN in mice, has already been found to be a tumor promotor (Sun et al., 2017; Wang et al., 2017; Zhang et al., 2017). Therefore, it is advantageous to investigate neuroprotective agents that not only lack a pro-tumorigenic potential, but also possess beneficial anticancer properties. Additionally, understanding the exact underlying mechanisms that contribute to CIPN is essential. Several processes are implicated in the pathogenesis of CIPN, including oxidative stress, dorsal root neuronal apoptosis, altered calcium homeostasis, mitochondrial dysfunction, and axonal degeneration leading to disturbed neuronal transport (Colvin, 2019). Moreover, the ability of chemotherapeutic agents to cause neurotoxicity via inflammatory processes has recently emerged as a fundamental element in the development of CIPN (Fumagalli et al., 2021). Subsequently, the investigation of immunomodulatory drugs for CIPN is now of great interest. Finally, despite that CIPN is more frequently reported in female human patients receiving cancer chemotherapy, preclinical evidence show no gender contribution to development of CIPN in murine models (Carpenter et al., 2021). However, our data would benefit from confirmation of rapamycin’s neuroprotective effect in female mice.

Our work provides data on the utility of rapamycin, an established immunomodulatory agent with anticancer effects, to propagate neuroprotection in a model of cisplatin-induced peripheral neuropathy. While cisplatin clearly induced neuronal damage accompanied by the development of thermal hypersensitivity, pre-treatment with rapamycin protected animals from exacerbated pain in cisplatin-treated groups. Response of rodents to thermal stimulators has been previously demonstrated in several studies based on thermal tests (hot plate, cold plate, plantar test, and tail flick test) (Deuis et al., 2017). Rapamycin prolonged the latency time by which animals reacted to the thermal inducers in the cisplatin-treated group as shown in Figure 1. These data are in agreement with previous studies showing that rapamycin has neuroprotective properties in other models of neuronal damage (Asante et al., 2010; Wang et al., 2016). In addition, rapamycin still exerts its effect (Figure 2) even when cisplatin was given acutely at higher dose (10 mg/kg), indicating that rapamycin is a potent neuroprotective agent. Several reports have indicated that some anticancer agents including cisplatin induce cell death in neuronal cultured from mice (Üstün et al., 2018). Thus, we intended to expand our experiments to include an ex vivo model. Consistent with our animal experiments, rapamycin has remarkably reduced apoptosis after exposure to cisplatin in a time-dependent manner (Figure 3). Taken together, rapamycin obviously attenuated the cisplatin-induced neurotoxicity both in vivo and ex vivo.

The neuroprotective effect of rapamycin in in this work can be attributed to several mechanisms. Recent studies showed that the neuroprotective effect of rapamycin is dependent on mTOR pathway blockade (Wang et al., 2016), whereas others pointed toward a reduction in the secretion of a spectrum of pro-inflammatory cytokines (Duan et al., 2018). Here, we sought to investigate whether the neuroprotective action of rapamycin is associated with modulation of systemic inflammation. Interestingly, we found that cisplatin significantly increased overall systemic inflammation, i.e., IL-17A expression in CD8 + T cells, whereas pre-treatment of mice with rapamycin remarkably reversed the cisplatin-induced effects on IL-17A expression (Figure 3). Earlier studies have shown an important role of IL-17A in causation of chemotherapy-induced neuropathic pain (Yao et al., 2016; Luo et al., 2019). Furthermore, reports have indicated that mTOR pathway is involved in mediating IL-17A production (Gulen et al., 2010; Nagai et al., 2013; Raïch-Regué et al., 2015; Chen et al., 2020), which is considered as a neurotoxic cytokine (Li et al., 2007; Kebir et al., 2007; Bǎlaşa et al., 2013; Debnath and Berk, 2014). IL-17A is a pro-inflammatory cytokine that is involved in neuroinflammatory and neuropsychiatric disorders such as autism, depression, and multiple sclerosis. It has been shown to be predominantly expressed by T cells such as CD8 + /CD4 + T cells. In the current study, IL-17A + immunostaining in CD8 + T/CD4 + T cells was not significantly different among different groups. However, IL-17A + immunostaining in immune cells other than CD8 + T cells was found be increased in cisplatin group which was significantly downregulated by rapamycin treatment. This observation suggests that rapamycin likely reduced expression of IL-17A in immune cells other than CD8 + T cells. Immune cells other than CD8 + /CD4 + T cells which express and secrete IL-17A include gamma delta T cells, NK cells, B cells, and granulocytes. Rapamycin-mediated reduction in IL-17A expression may occur in these immune cells. Future studies are required to ascertain which specific immune cells are affected by rapamycin treatment. Overall, our data point toward the possibility of IL-17A involvement in chemotherapy-induced neuropathic pain.

In addition to its immunomodulatory effects, rapamycin appears to decrease neuronal cell death. In our work, rapamycin decreased the expression of markers of apoptosis and senescence in response to cisplatin (Figure 4). Caspase-3 activity and the levels of PUMA, p53 and p21^Cip1 were found to be elevated when cells were exposed to cisplatin alone indicating genotoxic stress. In line with our findings, a recent paper by Hui et al. (2022) indicated that caspase 3 induction is necessary to for cisplatin-induced apoptosis in a neurotoxicity model and is considered a reliable marker for neuronal cell death (Hui et al., 2022). Several other studies unveiled the significance of the pro-apoptotic gene (PUMA) in neurodegeneration (Maor-Nof et al., 2016; Simon et al., 2016). Thus, it is of value to assess whether PUMA induction and caspase 3 activation are linked to cisplatin-induced neuroapoptosis. However, we encourage that future studies of cisplatin-induced neurotoxicity include cell viability assays along with more biomarkers of apoptosis such as annexin V staining and TUNEL assay. Recent evidence has suggested that cellular senescence plays a significant role in the development of CIPN. The first report by Acklin et al. (2020) showed that primary cultured DRG neurons isolated from adult C57BL/6 mice exposed to cisplatin ex vivo have upregulated senescence-associated-β-gal staining, a hallmark of senescence. Moreover, cisplatin treatment resulted in increased cytoplasmic staining of both p16^INK4a, a canonical marker of senescence, and HMGB1 of primary DRG neurons (Acklin et al., 2020). In peripheral neuropathy, primary DRG neuronal culture or DRG organotypic cultures are often used to perform in vitro experiments due to the high susceptibility of DRG neurons against cisplatin toxicity (Haberberger et al., 2020). However, like every model, these also have a downside, which is the heterogeneous neuronal population, complicating in some cases the drawing of conclusions. Besides, the neurotoxic effects of cisplatin-chemotherapy are not limited to the periphery, but also severely affect CNS neurons, giving rise to the term “chemobrain” (Chiu et al., 2017). Furthermore, the p53-dependent apoptotic neuronal death is a mechanism shared among neurons, regardless of whether they are in the central or peripheral nervous system (Krukowski et al., 2015; Chiu et al., 2017; Maj et al., 2017). Therefore, we chose to perform our experiments on the less heterogeneous primary cortical neuronal cell culture as the most suitable model to test our hypothesis, which has been also used to study chemotherapy induced peripheral neuropathy (Lehmann et al., 2020). These observations of senescence were extended to an in vivo CIPN model by Calls et al. (2021) who indicated that, in cisplatin treated BALB/c mice, which developed CIPN in 14 weeks of treatment, DRGs exhibited senescence-like features including: positive SA-β-gal staining, large, fused mitochondria, and accumulating lipofuscin granules (Calls et al., 2021). Moreover, single-cell RNAseq of DRG’s sensory neurons revealed significant upregulation of Cdkn1a which encodes for p21^Cip1 in agreement with our findings (Calls et al., 2021). Consequently, the removal of cisplatin-induced senescent cells reversed, to an extent, behavioral features of CIPN. While these cells were eliminated successfully by senolytic drugs (agents that selectively kill senescent cells), the use of senomorphics (agents that modulate the inflammatory phase of senescence) is encouraged (Carpenter et al., 2021). Accordingly, in our study, rapamycin can be hypothesized to have exerted its neuroprotective effect through its senomorphic potential, which was marked by the reduction of the senescence marker p21^Cip1 and the inflammatory mediator IL-17A (Laberge et al., 2015; Xu et al., 2021).

In conclusion, rapamycin showed effectiveness against cisplatin-induced peripheral neuropathy. Our behavioral analysis revealed the ability of rapamycin to reduce hypersensitivity to thermal inducers even at high doses. Also, rapamycin significantly decreased caspase activity, a marker of apoptotic cell death, in cisplatin-treated neurons. Furthermore, the increase of pro-inflammatory cytokine expression, IL-17A, was seen to be associated with cisplatin use, and its expression was reversed by pre-treatment with rapamycin. Further studies are needed to elaborate more about the involvement of inflammatory signals in chemotherapy-induced neuropathic pain. Collectively, addition of rapamycin with neuropathic pain-inducing anticancer agents might be beneficial to cancer patients and could improve cancer therapy outcomes.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This animal study was reviewed and approved by Dr. Musaad Ali Alshammari The Chairman of the Research Ethics Sub-Committee on Living Creatures.

FA-A was the fellow while MoA was the principal investigator of the project. MoA, MuA (physician), and TS developed the idea behind the project. MiA, MeA, and AA partially did the experimental design and data analysis. FA was an expert in neuroscience research and conceptually contributed to parts related to neuroscience. AN and SA performed the studies relating to neuroinflammation. RL was involved heavily in experiments relating to apoptosis and senescence. MoA and FA-A contributed to writing, performing experiments, data collection, and analysis. All authors contributed to the article and approved the submitted version.